Isolation and purification of recombinant antigens from Leptospira sp towards the development an of immunodiagnostic assays.
Silva ED1, Medeiros MA1, Esteves G S1, Cunha EC1, Amaral IC1, Ferreira AG P1, Nascimento HJ1, Silva Junior J G1,3 and Ko A2,4
1Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos), Fundação Oswaldo Cruz/MS; 2Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz/MS; 3Universidade Federal Rural do Rio de Janeiro (UFRRJ); 4Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University.
Leptospirosis, a bacterial infection that is transmitted through contact with the urine of animal reservoirs, has emerged to become a major public health problem in urban centers of Brazil and other developing countries. Effective treatment of leptospirosis depends on prompt and timely diagnosis in the early-phase of illness. However, early-phase leptospirosis may be recognized or mis-diagnosed as dengue or other causes of acute febrile illness because of its non-specific clinical presentation. Whole Leptospira-based ELISA (Enzyme linked immunosorbent) and microagglutination test (MAT) are standard tests, but have sensitivities of <70% in detecting early-phase leptospirosis. We have focused on developing Leptospira recombinant protein-based serologic tests, in ELISA formats, as a potential alternative for a test with improved performance characteristics. Firstly, a total of three recombinant fragments of Leptospira Ig-like protein, Lig ANI, LigBrep and LigBNI, were cloned and expressed in E. coli BL21(DE3) and by purified Affinity chromatography (Ni-IMAC). We developed an IgM ELISA using the three purified recombinant proteins and evaluated this assay with acute and convalescent serum samples from 29 MAT-confirmed leptospirosis patients and serum samples from 37 healthy blood bank donors from the city of Salvador, Brazil. The sensitivity and specificity for each of the recombinant protein-based IgM ELISA was the following: 77% sensitivity during acute-phase leptospirosis; 89% sensitivity during convalescent phase leptospirosis and 97% specificity for the LigBNI-based ELISA; 65% sensitivity during acute-phase leptospirosis; 78% sensitivity during convalescent phase leptospirosis and 95% specificity for the LigANI-based ELISA; and 69% sensitivity during acute-phase leptospirosis; 56% sensitivity during convalescent phase leptospirosis and 97% specificity for the LigBrep-based ELISA. These findings indicate that recombinant Lig protein-based ELISA may serve as an approach to developing diagnostic tests with improved sensitivity to identify leptospirosis in the initial stage of illness. Higher sensitivity may be achieved by defining the seroreactive epitopes and formulating kits with mixed cocktails of recombinant fragments.
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